Nuclear staining
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Dear all, I have to detect Doxorubicin in frozen tissue tumors from
treated vs untreated mice. I counterstained nuclei either with YOYO-1 after
permeabilization with 0.1% Triton or with SYBRGreen-1 with no permeabilization.
I set up sequential acquisition method with confocal BioRad2000
system as follows: PMT1 Ex= 488; Em = BP 515/30. PMT2 Ex= 488; Em = BP 600/50. Could someone explain me why I still detect a strong signal with
PMT2, even though there is the emission BP filter, in both cases (YOYO1 or SYBR
green)? Given that the signal is present also in untreated samples, it can be
due only to nuclear staining, can’t it? Thanks, Patrizia ....................................................................... Dr. Patrizia
Casalini Molecular Biology
Unit Experimental
Oncology Dept. Fondazione IRCCS Istituto
Nazionale dei Tumori Via Venezian, 1. 20133 Milano - Italy Phone: +39 02 23902563, Fax: +39 02
23903073 E-mail: [hidden email] AVVISO IMPORTANTE La presente comunicazione, che potrebbe contenere
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Kilgore, Jason |
Re: Nuclear staining - Vendor Response
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** VENDOR RESPONSE ** Dear Patrizia, Are you certain this is not due to
autofluorescence contributed by the cells or the doxorubicin? If you haven’t
yet, I would suggest trying to image a no-dye control and see if it gives off a
fluorescent signal at that wavelength. Jason Jason A. Kilgore Molecular Probes Tech Support From: Confocal
Microscopy List [mailto:[hidden email]] On Behalf Of Casalini Patrizia Dear all, I have to detect Doxorubicin in frozen tissue tumors from
treated vs untreated mice. I counterstained nuclei either with YOYO-1 after
permeabilization with 0.1% Triton or with SYBRGreen-1 with no permeabilization.
I set up sequential acquisition method with confocal
BioRad2000 system as follows: PMT1 Ex= 488; Em = BP 515/30. PMT2 Ex= 488; Em = BP 600/50. Could someone explain me why I still detect a strong signal
with PMT2, even though there is the emission BP filter, in both cases (YOYO1 or
SYBR green)? Given that the signal is present also in untreated samples, it can
be due only to nuclear staining, can’t it? Thanks, Patrizia ....................................................................... Dr. Patrizia
Casalini Molecular Biology
Unit Experimental
Oncology Dept. Fondazione IRCCS Istituto
Nazionale dei Tumori Via Venezian, 1. 20133 Milano - Italy Phone: +39 02 23902563, Fax: +39 02
23903073 E-mail: [hidden email] AVVISO IMPORTANTE La presente comunicazione, che potrebbe contenere
informazioni riservate e/o protette da segreto professionale, è indirizzata
esclusivamente ai destinatari della medesima qui indicati. Ogni informazione
qui contenuta, che non sia relativa alla nostra
attività caratteristica, deve essere considerata come non inviata. Nel caso in
cui abbiate ricevuto per errore la presente comunicazione, vogliate
cortesemente darcene immediata notizia, rispondendo a questo stesso indirizzo di e-mail, e poi procedere alla cancellazione di questo
messaggio dal Vostro sistema. E' strettamente proibito e potrebbe essere fonte
di violazione di legge qualsiasi uso, comunicazione, copia o diffusione dei
contenuti di questa comunicazione da parte di chi la abbia
ricevuta per errore o in violazione degli scopi della presente. Ricordiamo che
la tecnologia di trasmissione utilizzata non consente di garantire
l’autenticità del mittente né l’integrità dei dati IMPORTANT NOTICE This
communication, that may contain confidential and/or legally privileged
information, is intended solely for the use of the intended addressees. Every
information or advice contained in this communication is subject to the terms
and conditions provided by the agreement governing the engagement with such a
client. If you have received this communication in error, please notify us
immediately by responding to this email and then delete it from your system.
Any use, disclosure, copying or distribution of the contents of this
communication by a not-intended recipient or in violation of the purposes of
this communication is strictly prohibited and may be unlawful. The transmission
technology used to send this mail can’t grant neither the sender identity
nor the data integrity P Rispetta l'ambiente: se non ti è
necessario, non stampare questa mail. P Before printing, think about the
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Turns out that using "clear type" as the way to smooth edges of screen fonts
breaks the spectraviewer. This option is found in Display Properties--> appearance--> effects I dont know why -- just another Windows quirk, I suppose. Thanks to all of those that replied to my question. --aryeh -- Aryeh Weiss School of Engineering Bar Ilan University Ramat Gan 52900 Israel Ph: 972-3-5317638 FAX: 972-3-7384050 |
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Casalini Patrizia |
Re: Nuclear staining - Vendor Response
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In reply to this post by Kilgore, Jason
Dear Jason, Thank you for your response, actually I
did the control you mentioned and no contribution of autofluorescence has been
detected; about dxr, it is a fluorescent compound which emits in red. I performed nuclear staining with YOYO1
several times and had no problem, however the excitation in the red channel has
been usually done with HeNe (543 nm); the difference in this experiment was
only that to detect dxr, based on the United States patent 4,906,100, I need to
excite with Ar 488 (Abs peak around 470-500 nm) and to collect in the red
channel (em peak around 570nm). First I though it was an interaction
between dxr and DNA stains, however it seems not the case, since the signal is
still present also in dxr untreated samples. Probably there is something I miss, but I
can’t imagine what. Any suggestions are welcome! Patrizia From: Confocal
Microscopy List [mailto:[hidden email]] On Behalf Of Kilgore, Jason ** VENDOR RESPONSE ** Dear Patrizia, Are you certain this is not due to
autofluorescence contributed by the cells or the doxorubicin? If you
haven’t yet, I would suggest trying to image a no-dye control and see if
it gives off a fluorescent signal at that wavelength. Jason Jason A. Kilgore Molecular Probes Tech Support From: Confocal
Microscopy List [mailto:[hidden email]] On Behalf Of Casalini Patrizia Dear all, I have to detect Doxorubicin in frozen tissue tumors from
treated vs untreated mice. I counterstained nuclei either with YOYO-1 after
permeabilization with 0.1% Triton or with SYBRGreen-1 with no permeabilization.
I set up sequential acquisition method with confocal
BioRad2000 system as follows: PMT1 Ex= 488; Em = BP 515/30. PMT2 Ex= 488; Em = BP 600/50. Could someone explain me why I still detect a strong signal
with PMT2, even though there is the emission BP filter, in both cases (YOYO1 or
SYBR green)? Given that the signal is present also in untreated samples, it can
be due only to nuclear staining, can’t it? Thanks, Patrizia ....................................................................... Dr. Patrizia
Casalini Molecular Biology
Unit Experimental
Oncology Dept. Fondazione IRCCS Istituto
Nazionale dei Tumori Via Venezian, 1. 20133 Milano - Italy Phone: +39 02 23902563, Fax: +39 02
23903073 E-mail: [hidden email] AVVISO IMPORTANTE La presente comunicazione, che potrebbe contenere informazioni
riservate e/o protette da segreto professionale, è indirizzata esclusivamente
ai destinatari della medesima qui indicati. Ogni informazione qui contenuta,
che non sia relativa alla nostra attività
caratteristica, deve essere considerata come non inviata. Nel caso in cui
abbiate ricevuto per errore la presente comunicazione, vogliate cortesemente
darcene immediata notizia, rispondendo a questo stesso indirizzo di e-mail, e poi procedere alla cancellazione di questo
messaggio dal Vostro sistema. E' strettamente proibito e potrebbe essere fonte
di violazione di legge qualsiasi uso, comunicazione, copia o diffusione dei
contenuti di questa comunicazione da parte di chi la abbia
ricevuta per errore o in violazione degli scopi della presente. Ricordiamo che
la tecnologia di trasmissione utilizzata non consente di garantire
l’autenticità del mittente né l’integrità dei dati IMPORTANT NOTICE This
communication, that may contain confidential and/or legally privileged
information, is intended solely for the use of the intended addressees. Every
information or advice contained in this communication is subject to the terms
and conditions provided by the agreement governing the engagement with such a
client. If you have received this communication in error, please notify us
immediately by responding to this email and then delete it from your system.
Any use, disclosure, copying or distribution of the contents of this
communication by a not-intended recipient or in violation of the purposes of
this communication is strictly prohibited and may be unlawful. The transmission
technology used to send this mail can’t grant neither the sender identity
nor the data integrity P Rispetta l'ambiente: se non ti è
necessario, non stampare questa mail. P Before printing, think about the
environment |
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RICHARD BURRY |
Re: Nuclear staining - Vendor Response
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Patrizia
According to Hutun in Journal of Fluorescence 2004 14(2);217-222, the excitation spectra of doxorubicin is broad, including not only 488nm but also 543 and even a little at 405 nm. Emission is highest at 590 nm. We are using the green HeNi (543 nm) and the 590 nm emission filter. I do not know if this will help with the background, but try it! Dick ----- Original Message ----- From: Casalini Patrizia <[hidden email]> Date: Friday, January 23, 2009 3:56 am Subject: Re: Nuclear staining - Vendor Response To: [hidden email]
Richard W. Burry, Ph.D. Department of Neuroscience, College of Medicine Campus Microscopy and Imaging Facility, Director The Ohio State University Associate Editor, Journal of Histochemistry and Cytochemistry 277 Biomedical Research Tower 460 West Twelfth Avenue Columbus, Ohio 43210 Voice 614.292.2814 Cell 614.638.3345 Fax 614.247.8849 |
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